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. 2020 Aug 17;11:4132. doi: 10.1038/s41467-020-17952-5

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a GUARD RNA performance for Cas9 and, (b) BE3. Base editing rates for BE (% of NGS reads with SNPs) or indel rates for Cas9 (% of NGS reads with indels) for a subset of off-target sites. Off-targets are labelled with an identifier, gene name (if genic), gRNA name, and the number of mismatches between the gRNA and the off-target site. Data are pooled from two independent experiments from cells treated with doxycycline. Each point represents a GUARD RNA and are compared to three non-targeting (NT) control RNAs. GUARD RNAs are classed as functional if they reduce off-target editing by at least two standard deviations from the mean of the NT controls. Data shown here is a subset of that presented in Supplementary Fig. 10. c Lentiviral plasmid screening reveals GUARD RNAs that are effective at endogenous genomic loci. (Left panel) indel rates from GUARD RNA screening of EMX1 gRNA off-target site MFAP1, highlighting non-functional GUARD RNA (v1) and functional GUARD RNA (v2). Screening data shown here is a subset of that presented in Supplementary Fig. 10. (Right panels) indel rates from amplicon sequencing of on-target and off-target endogenous loci from Cas9-expressing HEK293 cells transfected with EMX1 gRNA and GUARD RNA v1, GUARD RNA v2 or a NT control (all at 25 nM). Data represent mean of two independent experiments with symbols representing each replicate. d GUARD RNAs can mediate base editing at position 1 and 2 within the 15-nt GUARD RNA. GUARD RNAs were grouped according to whether they had a cytosine at each nucleotide position or not, and P values were generated by comparing the % of NGS reads with SNPs between GUARD RNA groups with an unpaired, two-tailed student’s t-tests. e Functional GUARD RNAs predominantly have NGG PAMs. The proportion of functional GUARD RNAs from the Cas9 screen that had NGG PAMs versus NAG PAMs was compared using a two-sided Fisher’s exact test. f Model depicting important parameters for GUARD RNA design. For GUARD RNAs that reduce Cas9 off-target editing, reducing distance from the gRNA, having an NGG PAM, binding to the same DNA strand as the gRNA, increased GC-content and high concentration in the cell will all increase the effectiveness of CRISPR GUARD. For BE3, the presence of cytosines at position 1 or 2 is to be avoided for 15-nt GUARD RNAs to prevent editing. Source data are provided as a Source Data file.